We aim to determine crystal structures of proteins and protein-protein complexes that function in the release of HIV-1 particles from host cells. This project builds on preliminary results from the Sundquist laboratory that demonstrate a role for the vacuolar protein sorting (Vps) pathway in budding of HIV-1 and other enveloped viruses. Computational and yeast two hybrid approaches are being used to identify relevant interactions, which are then confirmed and quantified by Biacore biosensor analyses of recombinant proteins, and validated in a biological context using RNA interference. For example, yeast two hybrid analysis indicated an interaction between the cellular Vps protein TsglOl and the p6 domain of the HIV-1 Gag protein. The biosensor was then used to quantitate the interaction and localize the interacting residues to the (7)PTAP(lO) motif within p6. Finally, depletion of cellular Tsgl 01 (and other Vps proteins) by small interfering RNA arrested HIV-1 budding at a late stage, and budding was rescued by reintroduction of TsglOl, formally demonstrating the involvement of the Vps pathway in HIV budding. Despite the importance of the Vps pathway for the budding of HIV-1 and other enveloped viruses, as well as normal cellular functions, there is an almost complete lack of relevant atomic resolution structures. We therefore propose to pursue a high-throughput approach to determine crystal structures of TsglOl and complexes with its binding partners (Aim I ) , and structures of other Vps pathway proteins and their complexes (Aim 2). Unlike most "industrial style" structural genomics initiatives, however, our aims are tightly coupled to biochemical characterization and understanding of biological function. Thus far we have expressed and purified eight recombinant proteins, with many more currently in progress or planned. Although the results of crystallization trials are uncertain, we anticipate a significant success rate based upon our past success in determining the structures of HIV MA, CA and other proteins.